11-03: Quantitative analysis and engineering of fatty acid biosynthesis in E. coli

Fatty acids are central hydrocarbon intermediates in the biosynthesis of diesel from renewable sources. The ability to optimize the yield and productivity of fatty acid biosynthesis in a fermentation-friendly bacterium such as E. coli can open the door to microbial biodiesel production. By introducing multiple modifications into the E. coli genome, we have engineered a cell line that produces 4.5 g/L/day total fatty acid in a fed-batch fermentation. However, further enhancement of fatty acid biosynthesis in this cell line proved unpredictable. To develop a more reliable engineering strategy, a cell-free system was developed that enabled direct, quantitative investigation of fatty acid biosynthesis and its regulation in E. coli. Using this system, the strong dependence of fatty acid synthesis on malonyl-CoA availability was verified. In contrast, NADPH is unlikely to be limiting under most physiological conditions, but when that happens, low expression of a phosphite dehydrogenase mutant can be used to efficiently interconvert NADH and NADPH. Sharp maxima were observed in the dependence of fatty acid biosynthetic rates on E. coli thioesterase and acyl carrier protein concentrations, suggesting that both proteins play critical roles in allosteric control of fatty acid biosynthesis. Results from this cell-free system were confirmed via the generation and analysis of metabolically engineered strains of E. coli. Our quantitative findings highlight the enormous catalytic potential of the E. coli fatty acid biosynthetic pathway, and target specific steps for protein and metabolic engineering to enhance the catalytic conversion of glucose into biodiesel.